Molecular Dissection of Cytoplasmic Dynein

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DESCRIPTION (provided by applicant): The goal of this project is to characterize the mechanochemical properties of the molecular motor dynein using a structure- function approach in the model system, S. cerevisiae. This work is relevant to human disease because humans lacking functional axonemal dynein develop Kartagner syndrome, a disease resulting in chronic respiratory problems, infertility and mirror- image organ positioning. Cytoplasmic dynein is thought to be present in all eukaryotic cells and has multiple, essential roles in cell growth and division. Research in the dynein field has been hampered by the lack of a system in which mutant proteins can be made and analyzed in vitro. The first aim of this project is to develop in vitro assays to study the motile properties of affinity purified yeast cytoplasmic dynein. Such biophysical assays have been pioneered by the Vale lab and others. These assays will be used to test hypotheses described in the following two aims. Unlike other cytoskeletal molecular motors, dynein contains multiple nucleotide binding sites that may bind and hydrolyze ATP to produce the force that drives movement. The second aim of this project is to determine the relative contribution of each ATP binding site in dynein function by mutating conserved residues predicted to be involved in ATP hydrolysis. Finally, the role of dynein-associated proteins in dynein-mediated motility will be analyzed. One such associated protein is the Liscencephaly 1 protein, which, when defective causes a severe brain developmental disease in humans. As dyneins are evolutionarily distinct from the molecular motors kinesin and myosin, these studies represent a frontier in the motor field and may reveal novel mechanisms for motor protein function.